A. Field of the Invention
The present invention pertains to a method of generating the fluorescence of polyanionic fluorophores in low dielectric media with fluorescence activation produced by specific quaternary onium compounds. The chemistry may be used in solution phase or in a solid phase such as a low dielectric polymer coating, and when used as a coating can function as a fluorescent optical sensor material for analytes which are permeable to the coating and capable of modulating the fluorescence in a specific manner.
B. Description of Related Art
When the spectral properties of a colorimetric or fluorimetric dye are modulated by a specific analyte, in a concentration dependent manner, the dye is said to be capable of functioning as a transducer for said analyte. A large body of technology using colorimetric and fluorimetric dyes as transducers now exists. Historically these dyes have been used in solution analyses, but they are finding renewed applications as immobilized indicators on optical surfaces such as optical fibers, (W. Rudolf Seitz, CRC Critical Reviews in Anal. Chem., 1988, 19, 135).
Fluorescence based optical detection systems are preferred over those based on colorimetric or absorbance based detection schemes because fluorescence is more sensitive by several orders of magnitude since it is a zero background phenomenon. Also, more spectral discrimination may be incorporated into the analysis as one employs excitation and emission wavelengths as selectivity parameters. Additional selectivity is possible by measuring fluorescence phenomena other than direct intensity, such as fluorescence lifetimes and polarization. A technological advantage of fluorescence analysis is the compatibility of fluorescence with laser excitation.
The photophysical properties of fluorophores are intimately related to their molecular structure as well as to the nature and charge of any substituents on the central structure. Many of the fluorophores commonly used for optical sensors are negatively charged and are typically used in polar media.
The immobilization of fluorophores as transducers for optical sensors has been accomplished with a variety of methods, generally falling into four classes: 1) adsorption to a charged surface or charged polymer (Orellana, G., Moreno-Bondi, M. C., Segovia, E., Marazuela, M. C., Analytical Chemiststy, 64, 2210-2215, 1992), 2) entrapment in a reservoir or polymer with a cover membrane (Wolfbeis, O. S., Weis, L. J., Leiner, M. J. P., Ziegler, W. E., Analytical Chemistry, 60, 2028-2030, 1988), 3) covalent attachment as a monolayer to the substrate surface (Wolfbeis, O. S., Offenbacher, H., Kroneis, H., Marsoner, H., Mikrochimica Acta, I, 153-158, 1984) and 4) covalent attachment as a copolymer grafted to the surface (Munkholm, C., Walt, D. R., Milanovich, F. P., Klainer, S. M., Anal. Chem. 58, 1427-1430, 1986).
All of these methods have inherent limitations which may be a problem for particular applications. Anionic dyes that are immobilized by adsorption to a charged surface may have poor stability in aqueous samples and the dye will not be protected from other ionic constituents in the sample. Immobilization via entrapment in a liquid or gel reservoir phase requires the addition of a barrier membrane which serves to contain the dye while being permeable to the analyte of interest. For clinical applications this second membrane is typically a hydrophobic polymer. However, dual-phase optical sensors, prepared with two layers of incompatible material, often suffer from malfunction due to pinhole leaks and also exhibit decreased response times due to diffusional requirements through the hydrophobic membrane. Optical sensors with single layer coatings have been prepared with covalently immobilized dyes and dye-copolymers. However a covalent immobilization can be difficult to achieve while preserving the dye's intrinsic photophysical properties. Even with covalent immobilization of the transducer a barrier membrane will be necessary if the sensor requires that the dye be isolated in a particular internal electrolyte or buffer system.
If one could use a fluorophore in a hydrophobic or nonpolar polymer membrane the optical sensor components could be chemically partitioned from the aqueous sample. However, polyanionic dyes are extremely sensitive to the microenvironmental polarity of the solvent or solid matrix surrounding the fluorophore and many in this class are not fuorescent in nonpolar media. Fluorescein, which has a very high, pH dependent quantum yield in aqueous samples, has a nearly totally quenched fluorescence when observed in the nonpolar environment of a low dielectric solvent such as hexane or benzene, or when included in a hydropohobic polymer such as polystyrene. In such an environmentally quenched state the dye is not useful as an analytical reagent.